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Epithelial cell differentiation regulated by MicroRNA-200a in mammary glands.

Nagaoka K, Zhang H, Watanabe G, Taya K - PLoS ONE (2013)

Bottom Line: Here, we found that one miRNA, miR-200a, was relatively highly expressed in epithelial cell-rich organs such as mammary glands, lung, and kidney in mice.However, knockdown of miR-200a prevented increases in ß-casein and E-cadherin mRNA expression.Protein analysis revealed that E-cadherin signal was decreased and ZEB1 (a marker of EMT) was increased following miR-200a knockdown.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan. nagaokak@cc.tuat.ac.jp

ABSTRACT
Mammary gland epithelial cells undergo periodic cycles of proliferation, differentiation, and involution. Many studies have reported that miRNAs, which are small, non-coding RNAs, influence a variety of biological processes during posttranscriptional regulation. Here, we found that one miRNA, miR-200a, was relatively highly expressed in epithelial cell-rich organs such as mammary glands, lung, and kidney in mice. In mammary glands, miR-200a expression increased during mid-pregnancy through lactation; its expression was stimulated by lactogenic hormone treatment of mammary epithelial cells. Lactogenic hormone also induced the expression of milk protein ß-casein mRNA (a marker of cell differentiation) and E-cadherin mRNA (a marker of epithelial cells). However, knockdown of miR-200a prevented increases in ß-casein and E-cadherin mRNA expression. Protein analysis revealed that E-cadherin signal was decreased and ZEB1 (a marker of EMT) was increased following miR-200a knockdown. Finally, in a three-dimensional culture system modeling lumen-containing mammary ducts, miR-200a knockdown decreased the cavity formation rate and suppressed claudin-3 and par-6b expression, indicating reduced epithelial cell polarity. These observations suggest that miR-200a is important for maintaining the epithelial cell phenotype, which contributes to lactogenic hormone induction of cellular differentiation in mammary glands.

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Effect of miR-200a knockdown on 3D cavity formation.(A) EpH4 cells grown in an anchorage-independent manner were immunostained for E-cadherin and ZO-1. In the confocal images of the colonies, the green color indicates immunostaining for E-cadherin, red color indicates immunostaining for ZO-1, and blue color indicates DAPI. Scale bar  = 10 μm. (B) Approximately 60% of EpH4 cells formed cavities with polarized cells when cultured in a suspension with matrigel. Knockdown of miR-200a resulted in a significant (p<0.05) reduction in the cavity formation (48%). (C) EpH4 cells (control vs miR-200a-knocked down) grew on matrigel, and expression of cell polarity-related genes was analyzed by real-time PCR. (D) EpH4 cells grew on matrigel were immunostained for ZO-1, E-cadherin, Claudin-3 and ZEB1. Scale bar  = 10 μm. All experiments were repeated three times.
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pone-0065127-g005: Effect of miR-200a knockdown on 3D cavity formation.(A) EpH4 cells grown in an anchorage-independent manner were immunostained for E-cadherin and ZO-1. In the confocal images of the colonies, the green color indicates immunostaining for E-cadherin, red color indicates immunostaining for ZO-1, and blue color indicates DAPI. Scale bar  = 10 μm. (B) Approximately 60% of EpH4 cells formed cavities with polarized cells when cultured in a suspension with matrigel. Knockdown of miR-200a resulted in a significant (p<0.05) reduction in the cavity formation (48%). (C) EpH4 cells (control vs miR-200a-knocked down) grew on matrigel, and expression of cell polarity-related genes was analyzed by real-time PCR. (D) EpH4 cells grew on matrigel were immunostained for ZO-1, E-cadherin, Claudin-3 and ZEB1. Scale bar  = 10 μm. All experiments were repeated three times.

Mentions: Western blot analysis showed that ß-catenin (different marker of epithelial cell) as well as E-cadherin protein levels in DIP treatment were decreased by transfection with anti-miR-200a (Fig. 4A). Furthermore, results of the immunofluorescence analyses also showed that the E-cadherin signal decreased in anti-miR-200a-treated cells. A tight junction protein, ZO-1, did not change the protein level, but apical membrane localization was reduced and the signals were diffused throughout the cytoplasm (Fig. 4A and B). In contrast to these epithelial markers, ZEB1 protein (EMT marker) level was increased by the transfection with anti-miR-200a (Fig. 4A and Fig. 5D).


Epithelial cell differentiation regulated by MicroRNA-200a in mammary glands.

Nagaoka K, Zhang H, Watanabe G, Taya K - PLoS ONE (2013)

Effect of miR-200a knockdown on 3D cavity formation.(A) EpH4 cells grown in an anchorage-independent manner were immunostained for E-cadherin and ZO-1. In the confocal images of the colonies, the green color indicates immunostaining for E-cadherin, red color indicates immunostaining for ZO-1, and blue color indicates DAPI. Scale bar  = 10 μm. (B) Approximately 60% of EpH4 cells formed cavities with polarized cells when cultured in a suspension with matrigel. Knockdown of miR-200a resulted in a significant (p<0.05) reduction in the cavity formation (48%). (C) EpH4 cells (control vs miR-200a-knocked down) grew on matrigel, and expression of cell polarity-related genes was analyzed by real-time PCR. (D) EpH4 cells grew on matrigel were immunostained for ZO-1, E-cadherin, Claudin-3 and ZEB1. Scale bar  = 10 μm. All experiments were repeated three times.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672172&req=5

pone-0065127-g005: Effect of miR-200a knockdown on 3D cavity formation.(A) EpH4 cells grown in an anchorage-independent manner were immunostained for E-cadherin and ZO-1. In the confocal images of the colonies, the green color indicates immunostaining for E-cadherin, red color indicates immunostaining for ZO-1, and blue color indicates DAPI. Scale bar  = 10 μm. (B) Approximately 60% of EpH4 cells formed cavities with polarized cells when cultured in a suspension with matrigel. Knockdown of miR-200a resulted in a significant (p<0.05) reduction in the cavity formation (48%). (C) EpH4 cells (control vs miR-200a-knocked down) grew on matrigel, and expression of cell polarity-related genes was analyzed by real-time PCR. (D) EpH4 cells grew on matrigel were immunostained for ZO-1, E-cadherin, Claudin-3 and ZEB1. Scale bar  = 10 μm. All experiments were repeated three times.
Mentions: Western blot analysis showed that ß-catenin (different marker of epithelial cell) as well as E-cadherin protein levels in DIP treatment were decreased by transfection with anti-miR-200a (Fig. 4A). Furthermore, results of the immunofluorescence analyses also showed that the E-cadherin signal decreased in anti-miR-200a-treated cells. A tight junction protein, ZO-1, did not change the protein level, but apical membrane localization was reduced and the signals were diffused throughout the cytoplasm (Fig. 4A and B). In contrast to these epithelial markers, ZEB1 protein (EMT marker) level was increased by the transfection with anti-miR-200a (Fig. 4A and Fig. 5D).

Bottom Line: Here, we found that one miRNA, miR-200a, was relatively highly expressed in epithelial cell-rich organs such as mammary glands, lung, and kidney in mice.However, knockdown of miR-200a prevented increases in ß-casein and E-cadherin mRNA expression.Protein analysis revealed that E-cadherin signal was decreased and ZEB1 (a marker of EMT) was increased following miR-200a knockdown.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan. nagaokak@cc.tuat.ac.jp

ABSTRACT
Mammary gland epithelial cells undergo periodic cycles of proliferation, differentiation, and involution. Many studies have reported that miRNAs, which are small, non-coding RNAs, influence a variety of biological processes during posttranscriptional regulation. Here, we found that one miRNA, miR-200a, was relatively highly expressed in epithelial cell-rich organs such as mammary glands, lung, and kidney in mice. In mammary glands, miR-200a expression increased during mid-pregnancy through lactation; its expression was stimulated by lactogenic hormone treatment of mammary epithelial cells. Lactogenic hormone also induced the expression of milk protein ß-casein mRNA (a marker of cell differentiation) and E-cadherin mRNA (a marker of epithelial cells). However, knockdown of miR-200a prevented increases in ß-casein and E-cadherin mRNA expression. Protein analysis revealed that E-cadherin signal was decreased and ZEB1 (a marker of EMT) was increased following miR-200a knockdown. Finally, in a three-dimensional culture system modeling lumen-containing mammary ducts, miR-200a knockdown decreased the cavity formation rate and suppressed claudin-3 and par-6b expression, indicating reduced epithelial cell polarity. These observations suggest that miR-200a is important for maintaining the epithelial cell phenotype, which contributes to lactogenic hormone induction of cellular differentiation in mammary glands.

Show MeSH
Related in: MedlinePlus